Isolation of ECP. The strain was cultured in. cytophaga broth (ANACKER and ORDAL, 1959) at 20 Ž for 2 days. Twelve mg in wet weight of the

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1 Bacterial gill disease (BGD) causes high mortality in salmonid hatcheries in many countries. Numerous filamentous bacteria cover the surface of the gill and their irritating effect results in fusion of gill filaments. Flavobacterium sp. which shows nei ther gliding motility nor swarming growth was reported as a pathogen of this disease by KIMURA et al. (1978) and WAKABAYASHI et al. (1980). Since this pathogen does not invade the host gill tissue it is suggested that the pathogen adheres to the surface of the gill epitherium and then at close quarters secretes some stimulant products to the gill. So the process of this disease may bear no relation to an endotoxin but have a deep relation to some ECP. The purpose of this study was to isolate ECP and to examine their pathological activities. An attention was paid to the phenomenon that the infectiousness of this pathogen decreased or completely disap peared by washing the bacterial cells. Bacterial strain and fish. Flavobacterium sp. BGD 7721 (ATCC 35035) isolated from diseased Yamame (Oncorhynchus masou) in Gunma Pre fecture in 1977 was used. Rainbow trout with a Isolation of ECP. The strain was cultured in cytophaga broth (ANACKER and ORDAL 1959) at 20 Ž for 2 days. Twelve mg in wet weight of the culture was inoculated on CGY-agar in a petri dish and incubated at 20 Ž for 4 days. CGY agar was composed of 0.5% Bacto Casiton 0.3% Gelatin 0.1% Yeast-Extract and 1.2% Bacto agar and the ph was adjusted at 7.2. The bacteria were collected from the surface of the agar and suspended in 50 mm trisbuffer with ph 7.2. In order to obtain the ECP the bacterial cells were violently washed in the following way. The bacterial suspension was con tinuously cooled in iced water and subjected to 30 seconds agitation by whirling blender (Kinematica GmbH). The agitated solution was centrifugated at rpm for 10 minutes. Since a preliminary experiment revealed that a small amount of ECP still remained on the cell surface after the first washing the sediment was resuspended in 50 mm trisbuffer and the washing procedure was repeated. The supernatant obtained from the first and second washing procedures were gathered and filtrated through 0.45ƒÊm milipore to exclude the bacte rial cell completely. To a volume of this filtrate 4

2 volumes of aceton was added and allowed to stand virtually identical with that described for RBC. at room temperature for 1 hour. The precipitates were collected by centrifugation at 9000 rpm for 10 minutes and dissolved in a small amount of distilled water. The ECP were collected by freeze-drying this solution. The amounts of protein and carbohy drate were determined by Lory's method and by the phenol-sulfate acid method. Fish-toxity. Fish-toxity of the ECP was exam ined with juvenile rainbow trout. Five aquaria (ABCDE) of 2 liter in volume containing 1 liter of sterilized water were prepared. Eight fish were transferred to each aquarium and water tempera ture was kept at 15 Ž. No ECP was added to aqarium A. 75 mg 200 mg 1000 mg and 2000 mg of Effects of washing on the bacteria. The washed bacteria were examined for survival rate and in fectivity. As for survival rate living bacteria in the suspension were counted before and after the wash ing. Infectivity of the washed cells was compared with that of the unwashed cells through waterborn infection. Juvenile rainbow trout were exposed for 1 hour to the suspension of the unwashed and washed bacteria in densities of and CFU/ml respectively. Water in the aquarium was thoroughly replaced by the distilled water 1 hour after the exposure and then the first fish samples were taken. The gill tissues were sampled weighed and ho mogenized with a given volume of sterilized dis tilled water. Serial 10-fold dilutions of the homog the ECP were added to aquaria B C D and E respectively. Water in each aquarium was changed for sterilized water containing the prescribed amount of the ECP everyday. Two fish were col lected from each aquarium on the 1st. 3rd. 5th. and 7th. day and their gills were observed histologi cally by light microscope and scanning electron microscope. Enzyme activity. Caseinase activity was deter mined by the method of JONSSON and MARTIN (1964) and gelatinase activity was determined by the method of GREEN and LARKS (1955). The measure ments of some other enzyme activities were con ducted with Api ZYM system (Asuka Junyaku). enates were plated onto CGY agar and colonies of Flavobacterium sp. were counted. The remain ing fish were kept in the aquaria at 15 Ž and the water was changed for distilled water everyday. The gills of these fish were also examined for viable bacterial counts 24 and 72 hours after the exposure. Hemolytic activity. Hemolytic.activity in the ECP was tested with sheep red blood cells (SRBC). The ECP were dissolved in PBS at a concentration of 4 mg/ml. One ml of 5% SRBC suspension in PBS was added to 2 ml of the ECP solution. The mixture was incubated at 37 Ž for 2 hours and Agglutinating activity. Hemagglutinating activ then allowed to stand at 5 Ž overnight. ities for red blood cells (RBC) from various ani mals were tested by the microtiter technique. Each RBC was washed three times with 0.15 M phosphate buffered saline (PBS) with ph 7.2 and made up to a 5% (v/v) suspension with PBS. The ECP was dis solved in PBS at a concentration of 4 mg/ml for use. Diluters and droppers for ml and U plate were used. The mixture was incubated at 37 Ž for 2 hours then at 5 Ž overnight. Next the ECP were boiled for 20 minutes and treated with Pronase E (Junseikagaku). Agglutinating ac tivity for formalin-killed bacteria cells was also examined. Flavobacterium sp. BGD 7721 was cul Endotoxin. Endotoxin in the ECP was exam ined with Shwartzman reaction. Escherichia coli was cultured in brain heart infusion broth (Nissui) at 37 Ž overnight. The broth culture was centri fugated at rpm for 10 minutes and the super natant was sterilized by filtration (0.45 ƒêm mili pore). The filtrate was diluted to one-twentieth. First a male rabbit weighing about 2.5 kg was subcutaneously injected with 1 ml of the ECP so lution in PBS in concentration of 1 mg/ml. After 24 hours the rabbit was intravenously injected with 3 ml of the diluted filtrate. tured on CGY-agar at 20 Ž for one week and the. collected cells were suspended in 0.3% formalin solution. After 12 hours the cells were washed three times in distilled water and 10 mg of cells were re suspended in distilled water. The method was Antigenicity. Antisera for the ECP and the washed formalin-killed bacterial cells were prepar ed by injecting rabbits subcutaneously with the antigens in Freund's complete adjuvant. The

3 Characteristics of Extracellular Products of Flavobacterium sp. 169 Table 1. Influence of agitation by whirling blender on infectivity of the bacteria A challenge test was done with water bone infection. Unagitated bacteria (BGD 7721) was used in aquarium (A) and agitated bacteria was used in aquarium (B).Table 2. Hemagglutin

4 170 Mitsuru OTOTAKE and Hisatsugu WAKABAYASHI Table 3. Other enzyme activities (by Api ZYM) A concentration of the ECP was 4 mg/ml with 0.85% NaCl solution. Fig. 1. The result of the Ouchterlony's double diffusion test. A: the 4 mg/ml ECP solution with PBS. B: anti ECP serum. C: anti washed formalin-killed bacteria cells serum. to exist because the rabbits showed neither in flamation nor necrosis on their skin. Antigenicity. The result of Ouchterlony's double diffusion test was shown in Fig. 1. Five precipi tational lines a b c d and e were observed. Line b and c appeared to be associated with antigens common to the ECP and the washed bacterial cells. However lines a d and c appeared to be formed by antigens which were specific to the ECP. Discussion KUDO and KIMURA (1983) reported a hyper plasia-inducing material isolated from Flavobacte rium sp. It was prepared by adding an equal vol ume of 90% phenol at Ž to the bacterial culture. Aceton was used in this study instead of phenole to obtain ECP from the solution be cause its reaction was milder than phenol and reserved more activities in the ECP. The hemagglutinins produced by bacteria are generally devided into two categories which are the fimbriae and the soluble protein agglutinin (OOISHI 1981). The fimbriae is known to be one of the most effective means for bacteria to adhere to the host cell (JONES 1977). However the agglutinin contain ed in the ECP was not identified as the fimbriae type 1 of E. coli (DUGUID 1955) because its activity was not prohibited by the addition of D-mannose. Although informations are insufficient it has been generally recognized that soluble protein agglu tinins are assumed to have an important role in bacterial adherence to the cells of the high class creatures (OOISHI 1981). As for the fact that hemag glutinating titer varies in large scale according to the deriver of RBC This agglutinin was similar to the soluble protein agglutinin produced by other bac teria. The bacterioagglutinin in the ECP seems to function advantageously when the bacteria form flora on the surface of the gills which are always exposed to water circulation. It is known that bac terial protease makes its propagation on tissue more easily by preventing the host's immune sys tem and at the same time it works directly upon the tissue which is to be resolved and digested (ARAI 1981). The ECP from Flavobacterium sp. were considered to have indispensable functions in the infection and the outbreak of the BGD because it contained hemagglutinin bacterioag glutin and pro tease. This hypothesis seems to be supported by the fact that the washed cells of Flavobacterium sp. lose their infectivity. Acknowledgements Thanks are due to the staff of Yamanashi Prefectural Fisheries Experimental Station for help with the experiments.

5 Characteristics of Extracellular Products of Flavobacterium sp. 171